Clostridium difficile (C. difficile) associated disease (CDAD) is a major source of morbidity and mortality, costing the United States healthcare system approximately $1.1 billion/year due to medication costs or surgery leading to prolonged hospital stays. In addition, hospitals are experiencing increased rates of this disease, with physicians documenting markedly increased relapse rates following treatment with metronidazole. Mortality rates due to C. difficile are increasing, and new strains produce higher levels of toxin that are resistant to antibiotics. Colonic infection with C. difficile can result in a spectrum of conditions that range from mild diarrhea to severe colitis with potentially life-threatening complications. Although elderly hospitalized patients receiving antibiotics are still the main group at risk of CDAC, an increase in CDAD in younger populations with no previous contact either with the hospital environment or with antibiotics is emerging. Furthermore, CDAD in specific populations that were previously at low risk, such as children and pregnant women, is increasing. Therefore, protecting those who are most at risk, including those exposed to peri-operative and or prolonged antibiotic use should be a priority and is of the utmost significance.
The gram-positive, anaerobic, spore-forming bacterium C. difficile is recognized as the major cause of hospital-acquired diarrhea (2008, Wilcox et al., J Antimicrob Chemother 62(2): 388-396; 2008, Gould et al., Crit Care 12(1): 203). While as many as 3% of healthy outpatient adults may be colonized with the organism, this rate increases dramatically following hospitalization. Since 2000, extensive publications have described resurgence in the rates and severity of C. difficile disease in North America and Europe (Gould et al., Crit Care 12(1): 203). These events have renewed interest in novel approaches to disease treatment and prevention, including toxin-specific vaccines. Disease results from the production of the major cytotoxins, Toxins A and B, whose clinical manifestations range from asymptomatic carriage to diarrhea, toxic megacolon, and death. Toxins A and B are 300 kD and 270 kD proteins, respectively, and are members of the large clostridial cytotoxin family which share homology with the hemorrhagic and lethal toxins of C. sordelli and the alpha toxin of C. novyi. These glucosyl transferases inactivate a variety of host proteins, including Rho, Rac, and Cdc42, ultimately leading to the ascribed pathologic and clinical changes. Toxin A is considered to be the principal mediator of C. difficile associated disease in animal models. This Toxin mediates epithelial disruption leading to cellular entry of Toxin B, also an enterotoxin. The Toxins stimulate IL-8 and MIP-2 secretion, and promote neutrophil recruitment (2005, Voth et al., Clin Microbiol Rev 18(2) 247-263). In addition, Toxin B strains (TcdA-TcdB+) have been shown to be more potent that Toxin A strains (TcdA+TcdB-) in causing mucosal necrosis and decreasing barrier function (2009, Rupnik et al., Nat Rev Microbiol 7(7) 526-536). These toxin-mediated events have renewed interest in novel approaches to disease treatment and prevention, including toxin-specific vaccines.
Recent studies highlight the influence of toxin-specific, host antibody responses on the outcome of C. difficile colonization and infection. It has been observed that patients with anti-Toxin A antibodies before colonization with C. difficile spores have been observed as having significantly lower risk of progression to active and severe disease. Once infected, patients who develop strong anti-Toxin antibody responses clear their disease with antimicrobial treatment and remain disease free. Over the past 10 years, several passive and active immunization strategies have been tested against C. difficile infection in animals. These studies have highlighted the importance of the carboxy-terminal receptor-binding domain (RBD) as containing key epitopes for neutralizing the activity of toxin A and toxin B. Finally, intravenous administration of IgG developed against the RBD is protective in mice and hamsters. The role of anti-toxin humoral immune responses in protection against C. difficile disease is further validated by numerous passive and active immunization studies followed by toxin challenge (2010, Leav et al., Vaccine 28(4): 965-969). Further, it has been shown that transcutaneous immunization with formalin-treated C. difficile toxin A (CDA) induces systemic and mucosal anti-CDA immune responses (2007, Ghose et al., Infect Immun 75(6): 2826-2832). However, clinical indications for immune based therapies against C. difficile have not been fully defined for passive immunization, as efficacy of pooled immunoglobulin preparations is not well established as adjunct therapy (2005, Aslam et al., Lancet Infect Dis 5(9): 549-557). Moreover, there are certain stability issues with toxoid-based vaccine platforms (1995, Torres et al., Infect Immun 62(12): 4619-4627) that may limit their effectiveness in the clinic. Such studies provide strong scientific rationale for development of alternative next generation optimized vaccines against C. difficile Toxins. To that end, promising next generation DNA vaccine technology that includes mucosal targeting adjuvants (2010, Kraynyak et al., Vaccine 28(8):1942-1951; 2010, Kutzler et al., Gene Ther 17(1): 72-82) and electroporation delivery methods has been shown to be effective in the HIV vaccine platform.
DNA-based immunization has a number of advantages that make it an attractive vaccine platform (2006, Hokey et al., Springer Semin Immunopathol 28(3): 267-279; 2008, Kutzler and Weiner, Nat Rev Genet 9(10): 776-788). First, it has an improved safety profile compared to live attenuated viruses, which may revert back to their virulent form or spread to unintended individuals. The following qualities of DNA-based vaccines and adjuvants support use of this platform over toxoid-based strategies: ease of manipulation, proven production techniques, stability, and the lack of a cold chain requirement. In addition, DNA vaccines have been shown to elicit humoral and cellular responses and confer protection in small and larger animal models (2008, Kutzler and Weiner, Nat Rev Genet 9(10): 776-788). Taken together, these advantages make DNA-based immunizations a desirable vaccine modality for C. difficile. Increasing the transfection efficiency of target cells through various physical delivery methods including Electroporation is an area that has had success in the field. Importantly, the DNA vaccine platform has shown promise and is currently in Phase I clinical trials testing highly optimized antigenic constructs, supporting the safety and potential of this platform (2009, Yin et al., Virology 393(1): 49-55; 2005, Kutzler et al., J Immunol 175(1): 112-123; 2007, Chong et al, Vaccine 25(26): 4967-4982). DNA vaccines have historically been associated with induction of cellular immune responses, although more recently, it has demonstrated that delivery of DNA vaccines using Electroporation can be used to induce robust humoral responses in addition to strong cellular responses. This includes the induction of neutralizing antibody titers in animal models for a number of diseases—including avian influenza (2008, Laddy et al., PLoS One 3(6): e2517; 2007, Laddy et al., Vaccine 25(16): 2984-2989), smallpox (2011, Hirao et al, J Infect Dis 203(1): 95-102), dengue (2009, Ramanathan et al., Vaccine 27(46): 6444-6453; 2009, Ramanathan et al, Vaccine 27(32): 4370-4380), and chikungunya (2011, Mallilankaraman et al., PLoS Negl Trop Dis 5(1): e928).
While numerous platforms have been studied as candidate C. difficile vaccines (2007, Ghose et al., Infect Immun 75(6): 2826-2832; 2011, Oberli et al., Chem Biol 18(5): 580-588; 2011, Pechine et al., FEMS Immunol Med Microbiol June 24; 2011, Permpoonpattana et al., Infect Immun 79(6): 2295-2302; 2011, Sandolo et al., Eur J Pharm Biopharm June 1; 2008, Ni Eidhin et al, FEMS Immunol Med Microbiol 52(2): 207-218; 2008, Taylor et al., Vaccine 26(27-28): 3404-3409; 2009, Gardiner et al., Vaccine 27(27): 3598-3604), the number of products in the clinical pipeline is lacking. Given the tremendous cost and increasing prevalence of C. difficile infections, there is thus a need in the art for an effective vaccine therapy which protects against C. difficile associated disease. The present invention addresses this unmet need in the art.